Cyclodipeptide Synthase Research Articles

A Comprehensive Overview of the Cyclodipeptide Synthase Family Enriched with the Characterization of 32 New Enzymes.

Cyclodipeptide synthases (CDPSs) use as substrates two amino acids activated as aminoacyl-tRNAs to synthesize cyclodipeptides in secondary metabolites biosynthetic pathways. Since the first description of a CDPS in 2002, the number of putative CDPSs in databases has increased exponentially, reaching around 800 in June 2017. They are likely to be involved in numerous biosynthetic pathways but the diversity of their products is still under-explored. Here, we describe the activity of 32 new CDPSs, bringing the number of experimentally characterized CDPSs to about 100. We detect 16 new cyclodipeptides, one of which containing an arginine which has never been observed previously. This brings to 75 the number of cyclodipeptides formed by CDPSs out of the possible 210 natural ones. We also identify several consensus sequences related to the synthesis of a specific cyclodipeptide, improving the predictive model of CDPS specificity. The improved prediction method enables to propose the main product synthesized for about 80% of the CDPS sequences available in databases and opens the way for the deciphering of CDPS-dependent pathways. Analysis of phylum distribution and predicted activity for all CDPSs identified in databases shows that the experimentally characterized set is representative of the whole family. Our work also demonstrates that some cyclodipeptides, precursors of diketopiperazines with interesting pharmacological properties and previously described as being synthesized by fungal non-ribosomal peptide synthetases, can also be produced by CDPSs in bacteria.

Global analysis of prokaryotic tRNA-derived cyclodipeptide biosynthesis

BackgroundAmong naturally occurring small molecules, tRNA-derived cyclodipeptides are a class that have attracted attention for their diverse and desirable biological activities. However, no tools are available to link cyclodipeptide synthases identified within prokaryotic genome sequences to their chemical products. Consequently, it is unclear how many genetically encoded cyclodipeptides represent novel products, and which producing organisms should be targeted for discovery.ResultsWe developed a pipeline for identification and classification of cyclodipeptide biosynthetic gene clusters and prediction of aminoacyl-tRNA substrates and complete chemical structures. We leveraged this tool to conduct a global analysis of tRNA-derived cyclodipeptide biosynthesis in 93,107 prokaryotic genomes, and compared predicted cyclodipeptides to known cyclodipeptide synthase products and all known chemically characterized cyclodipeptides. By integrating predicted chemical structures and gene cluster architectures, we created a unified map of known and unknown genetically encoded cyclodipeptides.ConclusionsOur analysis suggests that sizeable regions of the chemical space encoded within sequenced prokaryotic genomes remain unexplored. Our map of the landscape of genetically encoded cyclodipeptides provides candidates for targeted discovery of novel compounds. The integration of our pipeline into a user-friendly web application provides a resource for further discovery of cyclodipeptides in newly sequenced prokaryotic genomes.

A Six-Oxidase Cascade for Tandem C-H Bond Activation Revealed by Reconstitution of Bicyclomycin Biosynthesis.

As a commercial antibiotic, bicyclomycin (BCM) is currently the only known natural product targeting the transcription termination factor rho. It belongs to a family of highly functionalized diketopiperazine (DKP) alkaloids and bears a unique O-bridged bicyclo[4.2.2]piperazinedione ring system, a C1 triol, and terminal exo-methylene groups. We have identified and characterized the BCM biosynthetic pathway by heterologous biotransformations, in vitro biochemical assays, and one-pot enzymatic synthesis. A tRNA-dependent cyclodipeptide synthase guides the heterodimerization of leucine and isoleucine to afford the DKP precursor; subsequently, six redox enzymes, including five α-ketoglutarate/Fe2+ -dependent dioxygenases and one cytochrome P450 monooxygenase, regio- and stereoselectively install four hydroxy groups (primary, secondary, and two tertiary), an exo-methylene moiety, and a medium-sized bridged ring through the functionalization of eight unactivated C-H bonds.

A Six‐Oxidase Cascade for Tandem C−H Bond Activation Revealed by Reconstitution of Bicyclomycin Biosynthesis

AbstractAs a commercial antibiotic, bicyclomycin (BCM) is currently the only known natural product targeting the transcription termination factor rho. It belongs to a family of highly functionalized diketopiperazine (DKP) alkaloids and bears a unique O‐bridged bicyclo[4.2.2]piperazinedione ring system, a C1 triol, and terminal exo‐methylene groups. We have identified and characterized the BCM biosynthetic pathway by heterologous biotransformations, in vitro biochemical assays, and one‐pot enzymatic synthesis. A tRNA‐dependent cyclodipeptide synthase guides the heterodimerization of leucine and isoleucine to afford the DKP precursor; subsequently, six redox enzymes, including five α‐ketoglutarate/Fe2+‐dependent dioxygenases and one cytochrome P450 monooxygenase, regio‐ and stereoselectively install four hydroxy groups (primary, secondary, and two tertiary), an exo‐methylene moiety, and a medium‐sized bridged ring through the functionalization of eight unactivated C−H bonds.

Identification of the Biosynthetic Pathway for the Antibiotic Bicyclomycin.

Diketopiperazines (DKPs) make up a large group of natural products with diverse structures and biological activities. Bicyclomycin is a broad-spectrum DKP antibiotic with unique structure and function: it contains a highly oxidized bicyclic [4.2.2] ring and is the only known selective inhibitor of the bacterial transcription termination factor, Rho. Here, we identify the biosynthetic gene cluster for bicyclomycin containing six iron-dependent oxidases. We demonstrate that the DKP core is made by a tRNA-dependent cyclodipeptide synthase, and hydroxylations on two unactivated sp3 carbons are performed by two mononuclear iron, α-ketoglutarate-dependent hydroxylases. Using bioinformatics, we also identify a homologous gene cluster prevalent in a human pathogen Pseudomonas aeruginosa. We detect bicyclomycin by overexpressing this gene clusterand establish P. aeruginosa as a new producer of bicyclomycin. Our work uncovers the biosynthetic pathway for bicyclomycin and sheds light on the intriguing oxidation chemistry that converts a simple DKP into a powerful antibiotic.

Mutations of Residues in Pocket P1 of a Cyclodipeptide Synthase Strongly Increase Product Formation.

Expression of a cyclodipeptide synthase gene from Nocardiopsis prasina (CDPS-Np) in Escherichia coli resulted in the formation of cyclo-(l-Tyr-l-Tyr) (1) as the minor and cyclo-(l-Tyr-l-Phe) (2) as the major products. Site-directed mutagenesis revealed a strong influence on product accumulation of the amino acid residues in pocket P1. An 8-fold increase in product formation for 1 and 10-fold for 2 were detected in the double mutant T82V_Y196F compared with the wild type.

Unravelling the mechanism of non-ribosomal peptide synthesis by cyclodipeptide synthases.

Cyclodipeptide synthases form cyclodipeptides from two aminoacyl transfer RNAs. They use a ping-pong mechanism that begins with transfer of the aminoacyl moiety of the first aminoacyl tRNA onto a conserved serine, yielding an aminoacyl enzyme. Combining X-ray crystallography, site-directed mutagenesis and affinity labelling of the cyclodipeptide synthase AlbC, we demonstrate that the covalent intermediate reacts with the aminoacyl moiety of the second aminoacyl tRNA, forming a dipeptidyl enzyme, and identify the aminoacyl-binding sites of the aminoacyl tRNAs.

Specificity determinants for the two tRNA substrates of the cyclodipeptide synthase AlbC from Streptomyces noursei.

Cyclodipeptide synthases (CDPSs) use two aminoacyl-tRNA substrates in a sequential ping-pong mechanism to form a cyclodipeptide. The crystal structures of three CDPSs have been determined and all show a Rossmann-fold domain similar to the catalytic domain of class-I aminoacyl-tRNA synthetases (aaRSs). Structural features and mutational analyses however suggest that CDPSs and aaRSs interact differently with their tRNA substrates. We used AlbC from Streptomyces noursei that mainly produces cyclo(l-Phe-l-Leu) to investigate the interaction of a CDPS with its substrates. We demonstrate that Phe-tRNAPhe is the first substrate accommodated by AlbC. Its binding to AlbC is dependent on basic residues located in the helix α4 that form a basic patch at the surface of the protein. AlbC does not use all of the Leu-tRNALeu isoacceptors as a second substrate. We show that the G1-C72 pair of the acceptor stem is essential for the recognition of the second substrate. Substitution of D163 located in the loop α6–α7 or D205 located in the loop β6–α8 affected Leu-tRNALeu isoacceptors specificity, suggesting the involvement of these residues in the binding of the second substrate. This is the first demonstration that the two substrates of CDPSs are accommodated in different binding sites.

A tRNA-Dependent Two-Enzyme Pathway for the Generation of Singly and Doubly Methylated Ditryptophan 2,5-Diketopiperazines

A large number of bioactive natural products containing a 2,5-diketopiperazine (DKP) moiety have been isolated from various microbial sources. Especially tryptophan-containing cyclic dipeptides (CDPs) show great structural and functional diversity, while little is known about their biosynthetic pathways. Here, we describe the bioinformatic analysis of a cyclodipeptide synthase (CDPS)-containing gene cluster from Actinosynnema mirum spanning 2.9 kb that contains two putative DKP-modifying enzymes. We establish the biosynthetic pathway leading to two methylated ditryptophan CDPs through in vivo and in vitro analyses. Our studies identify the first CDPS (Amir_4627) that shows high substrate specificity synthesizing only one main product, cyclo(Trp-Trp) (cWW). It is the first member of the CDPS family that can form ditryptophan DKPs and the first prokaryotic CDPS whose main product constituents differ from the four amino acids (Phe, Leu, Tyr, and Met) usually found in CDPS-dependent CDPs. We show that after cWW formation a S-adenosyl-l-methionine-dependent N-methyltransferase (Amir_4628) conducts two successive methylations at the DKP-ring nitrogens and additionally show that it is able to methylate four other phenylalanine-containing CDPs. This makes Amir_4628 the first identified DKP-ring-modifying methyltransferase. The large number of known modifying enzymes of bacterial and fungal origin known to act upon Trp-containing DKPs makes the identification of a potent catalyst for cWW formation, encoded by a small gene, valuable for combinatorial in vivo as well as chemoenzymatic approaches, with the aim of generating derivatives of known CDP natural products or entirely new chemical entities with potentially improved or new biological activities.

Insights into the Generation of Structural Diversity in a tRNA-Dependent Pathway for Highly Modified Bioactive Cyclic Dipeptides

The nocazines are a newly defined family of antibacterial and cytotoxic cyclic dipeptides produced by different actinobacterial species. Here, we identify a nocazine biosynthetic gene cluster in Nocardiopsis dassonvillei and describe the elucidation of the biosynthetic pathway leading to the nocazine family members nocazine E and XR334. Diketopiperazine (DKP) formation is carried out by a tRNA-dependent cyclodipeptide synthase (CDPS) showing an unknown product profile, while tailoring of the DKP-scaffold is achieved through the combined and combinatorialaction of a cyclodipeptide oxidase and two distinctSAM-dependent O-/N-methyltransferases. Our results help to illuminate the biosynthetic logic resulting in the structural diversity of the nocazine family and set the stage for exploring the biological function of modified cyclic dipeptides as possible mediators of host-pathogen and host-parasite interactions.